Method of detection of carcinogenic human papillomavirus

ABSTRACT

High risk carcinogenic human types 16, 18 and 33 are distinguished from low risk human papillomavirus types 6 and 11 in a sample of human cervical tissue. A selected characteristic portion of the E6 region of the virus defined by specific oligonucleotide primers is amplified using a polymerase chain reaction. The presence or absence of the characteristic portion of the E6 region is detected by gel electrophoresis or using a labeled oligonucleotide probe.

This is a continuation of application Ser. No. 07/423,401 filed on Aug.25, 1989, now U.S. Pat. No. 5,783,412, which is a 371 of PCT/AU88/00047filed Feb. 24, 1988, claims the benefit thereof and incorporates thesame by reference.

TECHNICAL FIELD OF INVENTION

This invention relates to a method for the specific detection of the DNAof papillomaviruses in clinical samples. In particular, the test aims todifferentiate, in the shortest possible time, whether cells from theanogenital region contain types of papillomavirus that are associatedwith benign lesions. Such differentiation has important implications inpatient evaluation and follow-up.

BACKGROUND

Cancer of the cervix is the most common cancer in women (˜25% of allfemale cancer). Moreover, the incidence is increasing in younger women.Indeed, approximately 2% of routine cervical smears show abnormalcytology, indicating an epidemic. Such an epidemic is current in manywestern and developing countries. Sexual activity appears to be animportant predisposing factor in the epidemiology of cardinogenesis andprecancerous lesions. An early age of sexual intercourse andmultiplicity of sexual partners are associated statistically with anincreased risk of malignancy [Harris et al., Br. J. Cancer 42: 359-63,1980]. The consorts are often men with penile warts (“high risk males”),and a very high proportion (>90%) of cervical carcinoma tissue containdetectable DNA sequences for known varieties of the human papillomavirus(HPV). This supports a growing body of evidence implicating certaintypes of HPV as the sexually transmitted factor involved in thedevelopment of squamous-cell carcinoma of the cervix [zur Hausen et al.,Progr. Med. Virol. 30: 170-86, 1984; zur Hausen, Prog. Med. Virol. 32:15-21, 1985; zur Hausen, Cancer 59: 1692-6; Campion et al., Lancet 1:943-6, 1985]. The prevalence of cervical cancer and precancerous lesionsis becoming increasingly more common in younger women. Without treatmentit can be fatal, the death rate being ˜100 per million women per year inWestern countries. Fortunately, if detected at an early stage, effectivetreatment is available that can eliminate the fatal consequences.

The immediate management and subsequent follow-up of young women withabnormal cytological smears who still wish to bear children presentsmany problems. This has been compounded by uncertainty about theinterpretation of smears with features of papillomavirus infection(“kollocytes”) as well as dysplasia. Moreover, the current cytologicaltesting tool for cervical cancer screening, the Pap smear, has a falsenegative rate of ˜20%. Significant numbers of dysplastic lesions regressspontaneously, others fail to progress, while a few progress rapidly.Thus, from an ill-defined cloud of morphological abnormalitiesoccasional cancers develop. At present there is no clear way to predictwhether cancer will result if a Pap smear happens to be abnormal.Clinical examination of many of these patients has failed to find wartylesions (condylomata accuminata) on the external genitalia or, indeed,on the cervix itself. The more difficult procedure of colposcopy, afterthe application of 3% acetic acid, is, in fact, required, revealing thepresence of flat (“non-condylomatous”) warts (which are invisible to thenaked eye). These are the expected premalignant lesions.Histophathological progression of the wart to carcinoma in situ andfrank malignancy has been well described [e.g., Dyson et al., J. Clin.Path. 37: 126-31, 1984]. An increasingly prevalent problem is theoccurrence of invasive cancer within 3 years of a negative Pap smear[Berkowitz et al., Gynecol. Oncol. 8: 311, 1979; Holman et al., Med. J.Aust. 2: 597, 1981]. Whereas the presence of papillomavirus replicationmay be confirmed in cervical condylomata by detection of virus particlesor the group-specific antigen, neither particles nor antigen have,however, been found in squamous cell carcinoma tissue.

In contrast to the uncertainty and controversy that surrounds theinterpretation of tests based on morphology, the new techniques inmolecular biology can be utilised to bypass such problems and providemore objective information. By using nucleic acid hybridizationtechniques the viral DNA can be identified directly and at an earlierstage of infection. Indeed, using these approaches, HPV types have beenfound in both benign and premalignant lesions.

At present ˜50 types of the papillomavirus have been distinguished inhuman infection. Different ones infect different epithelial areas. Theparticular types of HPV that commonly infect the genital tract includethose assigned the numbers 6, 11, 16, 18 and several rarer types (31,33, 35, 39, 43 and 44). HPV6 [de Villlers et al., J. Virol. 40: 932-5,1981] and HPV11 [Gissmann et al., J. Virol. 44: 393-400, 1982; Gissmanet al., Proc. Natl. Acad. Sci. USA 80: 560-3, 1983; Dartmann et al.,Virology 151: 124-30, 1986] have been associated with benign condylomataaccuminata, the classical lesion of the anal and genital tract [Gissmannet al., J. Invest. Dermatol. 83: 26s-8s, 1984]. In contrast, HPV16[Durst et al., Proc. Natl. Acad. Sci. USA 80: 3812-5, 1983] and HPV18[Boshart et al., EMBO J. 3: 1151-7, 1984; Cole and Danos, J. Mol. Biol.193: 599-608, 1987] are more often detected in dysplastic flat lesionsof the vulva and cervix, and squamous carcinoma of the cervix and penis[Crum et al., Cancer 49: 468-71, 1982; Campion et al., Lancet i: 943-6,1985].

Thus the types of HPV that infect the anogenital area can be assigned totwo categories as follows:

1. “Low-risk”; HPV 6 and HPV11, with type 6 being the most common of allanogenital types.

2. “High-risk”; HPV16, HPV18, HPV31, HPV33, HPV35, HPV39 HPV43 andHPV44.

The frequency of occurence of the higher risk types is in decreasingorder. Thus, within the high risk category, HPV16 is most common(45-60%), HPV18 is next most common (20-30%) and the others are rarer,the last 4 being discovered only recently and reported in 1986 (totalfrequency for all of these rarer types is, collectively, ˜15%). Otherrarer types are likely to be discovered in due course.

In support of a role for HPVs in cervical cancer the following findingsare noteworthy:

(i) DNAs of known high risk HPVs have been detected in ˜90% of cervicaladenocarcinomas and squamous cell carcinomas [Zachow et al., Nature 300:771-3, 1982; Gissmann et al., 1984, ibid].

(ii) High risk HPV DNA has been found in metastases arising fromcervical tumours [Lancaster et al., Am. J. Obstet. Gynecol. 154: 115-9,1986].

(iii) Instead of being present in cells in the usual episomal form, DNAsof high risk HPVs have been found integrated into human genomic DNA[Schwartz et al., Nature 314: 111-4, 1985; Lehn et al., Proc. Natl.Acad. Sci. USA 82: 5540-4, 1985; Kreider et al., Nature 317: 639-41,1985; Matsukura et al., J. Virol. 58: 979-82, 1986; Schneider-Gadickeand Schwartz, EMBO J. 5: 2285-92, 1986; Di Luca et al., J. Gen. Virol.67: 583-9, 1986]. Such integration has been suggested to be necessaryfor malignant conversion of the cells, supported by findings ofintegration also in precarcinoma tissue [Shirasawa et al., J. Gen.Virol. 67: 2011-5, 1986].

(iv) The integration pattern usually interrupts or deletes specificregions of the HPV16 or 18 DNA, but consistently leaves intact the E6and E7 openreading frames (ORFs) ([Pater and Pater, Virology 145: 313-8,1985], which continue to express, at least in cell lines derived fromcervical carcinomas [Smotkin and Wettstein, Proc. Natl Acad. Sci. USA83: 4680-4, 1986; Androphy et al., EMBO J. 6: 989-92, 1987; Baker etal., J. Virol. 61: 962-71, 1987; Takebe et al., Biochem. Biophys. Res.Commun. 143: 837-44, 1987].

(v) A splice donor exists in the E6 ORF of HPV16 and 18 (but not HPV6and 11) which can result in the generation of an ORF which whentranslated resembles epidermal growth factor [zur Hausen, Lancet 489-91,1986].

(vi) Integration in cervical cell lines (HeLa, CaSki, SiHa, SW756, etc)is often near proto-oncogenes [Dürst et al., Proc. Natl. Acad. Sci. USA84: 1070-4, 1987; Popescu et al., Cytogenet. Cell Genet, 44: 58-62,1987; Popescu et al., J. Virol, 51: 1682-5, 1987; Shirasawa et al., J.Gen. Virol. 68: 583-91, 1987].

(vii) Such integration is associated with increased expression of c-mycand c-ras mRNA [Dürst et al., 1987, ibid; Shirasawa et al., 1987,ibid.), consistent with the suggestion that cis-activation of cellularoncogenes by HPV might be associated with malignant transformation ofcervical cells.

(viii) Human fibroblasts and keritanocytes can be transformed bytransfection with HPV16 [Pirisi et al., J. Virol. 61: 1061-6, 1987], ascan NIH 3T3 cells [Tsunokawa et al., Proc. Natl. Acad. Sci. USA 83:2200-3, 1986; Yasumoto et al., J. Virol. 57: 572-7, 1986].

(ix) Integration might disrupt genes coding for cellular interferingfactors: this may cripple the cells normal defense mechanism thatsuppresses uncontrolled growth and transcription of the virus [zurHausen, Lancet 489-91, 1986].

Whereas penile warts in males only very rarely result in cancer of thepenis, when transmitted to the cervix cancer is much more likely tofollow. About one-third of patients who have histologically-confirmedHPV infection of the cervix can be expected to develop cervicalintraepithelial neoplasia (CIN) within a year [Nashet al., Obstet.Gynecol. 69: 160-2, 1987]. The lag time between infection and cancer is,however, often 10-30 years. Thus the unique environment of the cervix,coupled with other factors, such as smoking [Trevathan et al., J. Am.Med. Ass. 250: 449-504, 1983], also contributed to the onset of thecancer. Damage of DNA by the latter, coupled with HPV's action to causecellular proliferation, may explain the onset of malignancy. Treatmentof women with precancerous lesions involves surgical extirpation of theaffected area. Moreover, it is now believed by many that treatmentshould also involve her infected male consort, in order to avoidreinfection and infection of other women by the man.

Since the cytological test used presently for routine screening ofcervical cells (Pap smear) is considered subjective and does not allowthe identification of the particular type of HPV in a lesion, we foreseethat the more specific approach of DNA—DNA hybridization for directviral detection will in due course be used routinely to supplement oreven replace cytology in primary screening.

In the clinical evaluation of a patient it is important to distinguishbetween those lesions harbouring the potentially carcinogenic(high-risk) types from those associated with the more benign (low-risk)types.

The evaluation of patient specimens for HPV infection has beenfacilitated by techniques of filter hybridization. Such techniques havebeen used to detect HPV DNA in cervical scrapes collected in parallelwith samples for routine cytology [Wagner et al., Obstet. Gynecol. 64:767-72, 1984; Wickenden et al., Lancet i: 65-7, 1984; Schneider et al.,Science 216: 1065-70, 1982]. In January 1985 a project was begun by Dr.Morris at the University of Sydney to develop a direct test for theanogenital types of HPV; with a particular aim of evaluating whether theinfection was by one of the high risk types of HPV or by one of the lowrisk types. The first publication describing a preliminary version ofthe test, which involved recombinant viral DNAs, is: B. R. Henderson, C.H. Thompson, B. R. Rose, Y. E. Cossart and B. J. Morris, “Detection ofspecific types of human papillomavirus in cervical scrapes, analscrapes, and anogenital biopsies by DNA hybridization”, Journal ofMedical Virology 12: 381-93, 1987. This paper was submitted early in1986. Since then the sensitivity of the recombinant DNA test has beenincreased 100-fold and further improvements are being made continually.Over 5,000 clinical specimens have been tested to date. These have beenmainly from Sydney S.T.D. clinics. The data so far has established theviability and usefulness of direct viral detection for determination ofthe presence and nature of HPV infection in cervical scrapes and otheranogenital specimens. Our other recent publications using this approachinclude: B. R. Rose, C. H. Thompson, A. M. McDonald, B. R. Henderson, Y.E. Cossart & B. J. Morris, “Cell Biology of cultures of anogenitalwarts”, British Journal of Dermatology 116: 331-22, 1987; B. J. Parker,Y. E. Cossart, C. H. Thompson, B. R. Rose & B. R. Henderson, “Theclinical management and laboratory assessment of anal warts”, MedicalJournal of Australia 147: 59-63, 1987; P. M. Katelaris, Y. E. Cossart,B. R. Rose, B. Nightingale, E. Sorich, C. H. Thompson, P. B. Dallas & B.J. Morris, “Human papillomavirus: The untreated male reservoir”, Journalof Urology, in press, 1988. Much other work has yet to be published.

For main strand DNA sequences of the most common anogenital HPV typessee as follows:

The sequence of HPV6b is given in Schwartz et al., EMBO J. 2: 2341-2348.

The sequence of HPV11 is given in Dartmann et al., Virology 151: 124-30,1986.

The sequence of HPV16 is given in Seedort et al., Virology 145: 181-5,1985.

The sequence of HPV18 is given in Matlashewski et al., J. Gen. Virol.67: 1909-16, 1986.

The sequence of HPV33 is given in Cole and Streeck, J. Virol. 58: 991-5,1986.

Principal of detection of specific viral DNA by hybridization

DNA is double-stranded. Each strand of DNA is a complementary ‘mirrorimage’ of the other. The DNA strands are held together by hydrogenbonding. Our techniques for detecting viral DNAs are based on theability of the unique sequence of nucleotides in a DNA strand to bondwith (‘hybridize’) to a sequence complementary to it. Thus, armed with aDNA sequence for all or a unique part of a papillomavirus type, it ispossible to use this as a ‘homing probe’ in order to detect the virus ina sample of cervical cells from a patient. The DNA for use as probe islabelled either with a radioactive isotope or nonradioactive label sothat it can be detected later. To increase the sensitivity of the testwe have utilized a method for amplification of the HPV DNA sequences inthe sample.

Background to approach used to amplify viral DNA

In order to increase the sensitivity of the detection technique we use amethod described originally for diagnosis of genetic diseases. This isknown as a ‘polymerase chain reaction’ (PCR). It is used to amplifyenzymatically a specific DNA sequence before hybridization withsynthetic oligonucleotide probe. A typical amplification factor is˜250,000 copies starting from one copy of viral DNA. Such an approachnot only increases sensitivity, but fulfills requirements ofspecificity, speed, simplicity, and amenability to nonradioactivedetection methods expected of a more versatile testing procedure. It isalso amenable to assembly as a kit and to automation, both of which wehave accomplished. The PCR technique is described in papers that dealwith prenatal diagnostic testing for specific genetic abnormalities[Sakii et al., Science 230: 1350-4, 1985; Sakii et al., Nature 324:163-6, 1986 ; Scharf et al., Science 233: 1076-8, 1986]. The PCRtechnique is the subject of the following: Australian Patent ApplicationAU-A-55322/86, Cetus Corp.” Process for Amplifying Nucleic AcidSequences”, U.S. Priority Date 28.3.85; Australian Patent ApplicationAU-A-55323/86, “Amplification and Detection of Target Nucleic Acid byHybridization Probe”, U.S. Priority Date 28.3.85.

Briefly, a small, unique portion of the HPV DNA sequence, ˜100-200 bplong, is amplified by the PCR procedure. In the examples a region in theE6 region has been chosen. However, any other region in the viral DNAmay also be chosen. The PCR step requires two ˜20 mer oligonucleotideprimers that flank the region to be amplified. One primer iscomplementary to the (+)-strand of a region of the DNA and the other iscomplementary to the (−)-strand. The annealing of primer to the(+)-strand of denatured sample viral DNA, followed by extension with,e.g., the Klenow fragment of Escherichia coli DNA polymerase, or otherenzymes that carry out a similar reaction, and deoxynucleotidetriphosphates results in the synthesis of a (−)-strand fragmentcontaining a ‘target’ sequence residing between the hybridization sitesof the primer. At the same time a similar reaction occurs with the otherprimer, creating a new (+)-strand. The principle of the method is shownin the following diagram.

Since these newly-synthesized DNA strands are themselves templates forthe PCR primers, repeated cycles of denaturation, primer annealing, andextension result in the exponential accumulation of the ˜100-200 bpregion defined by the primers. Next, the specific DNA is detected.Various means are possible for doing this. The amount of DNA producedmay be sufficient for direct visualization after electrophoresis on agel and staining.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the amplification of pure HPV16 DNA using PCR relativeto a standard restriction of pBR322 with HpAII.

FIG. 2 indicates the amplification of HPV16 DNA from wart biopsyrelative to a standard restriction of pBR322 with HpAII.

FIG. 3 indicates the hybridization of 32P-labeled HPV16/13 targetoligonucleotide probe with PCR amplified HPV 16 DNA from wart biopsy.

FIG. 4 indicates the amplification of pure HPV 16 recombinant viral DNAusing PCR (Lane 1) relative to standard DNA size markers (Lane 2 -pBR322 cut with HpAII; Lane 3 - pBR322 cut with SPP-1).

FIG. 5 indicates the hybridization of HPV16/33 oligonucleotide probewith HPV16 DNA taken from Lane-2 of FIG. 4.

FIG. 6 indicates the hybridization of HPV16/33 oligonucleotide probewith PCR amplified HPV16 DNA from anal wart biopsies.

FIG. 7 indicates that the HPV 16/33 oligonucleotide probe hybridizes tothe correct restriction fragments of unamplified biopsies (Lanes1,3,4,). FIG. 7 also indicates that the HPV16/33 oligonucleotide probenot hybridize to other HPV types (Lanes 5,6,7).

FIG. 8 indicates the hybridization of radioactively labeled HPV16/33oligonucleotide probe with PCR amplified DNA from cervical scrapes.

FIG. 9 indicates the amplification of the cervical scrape DNA used inFIG. 8 (Pt. cervical scrape) using PCR.

FIG. 10 indicates the hybridization of radioactively labeled HPV16/33oligonucleotide probe with PCR amplified Pt. Cervical scrape.

FIG. 11 indicates the hybridization of alkaline phosphatase labeledHPV16/33 probe with the PCR amplified Pt. Cervical scrape.

FIG. 12 indicates the PCR amplification of HPV6/11 DNA from variousbiopsies and scrapes.

FIG. 13 indicates the hybridization of radioactively labeled HPV6/11oligonucleotide probe with PCR amplified DN from cervical scrape(Lane-4) and vaginal scrape (Lane-5).

FIG. 14 indicates the PCR amplification of HPV6/11 DNA from various wartbiopsies.

FIG. 15 indicates the PCR amplification of HPV6/11 DNA from Hn biopsy.

FIG. 16 indicates that the PCR primers for different HPV types can beused in the same reaction mixture to generate amplification productsunique for each HPV type. HPV6/11 and HPV16/33 primers were used in thesame reaction mixture. Lane 2 shows HPV16/33 amplification product andHPV6/11 amplification product.

FIG. 17 indicates the PCR amplification of HPV16/33 DNA from varioussources.

FIG. 18 indicates the hybridisation of alkaline phosphatase probes withthe HPV16/33 DNA from FIG. 17.

FIG. 19 indicates the hybridisation of radioactively labelled probeswith HPV16/33 DNA from FIG. 17.

FIG. 20 is a dot blot indicating the hybridisation of radioactivelylabelled probes with various HPV types.

FIG. 21 indicates that heat stable polymerase from Thermus Aquaticus isable to be used to amplify HPV DNA.

FIG. 22 indicates that heat stable polymerase from Thermus Aquaticus isable to amplify HPV16 DNA from biopsies.

FIG. 23 indicates that HPV DNA from diluted biopsies was amplified usingPCR.

FIG. 24 indicates the amplified DNA from the diluted hybridising withradioactively labelled HPV6/11 oligonucleotide probe.

FIG. 25 indicates the amplified DNA from the diluted biopsieshybridising with radioactively labelled HPV16/33 oligonucleotide probe.

FIG. 26 indicates unamplified HPV16, HPV33 and biopsies, run on a geland stained with ethidium bromide.

FIG. 27 indicates the hybridisation of HPV16/33 oligonucleotide probewith unamplified HPV16, HPV18 and biopsies.

FIG. 28 indicates hybridisation of alkaline phosphatase labelledHPV16/33 oligonucleotide probe with HPV16 DNA.

FIG. 29 indicates the amplification of HPV18 DNA using PCR.

DISCLOSURE OF THE INVENTION

In particular, the present invention provides a method of detection ofcarcinogenic human papillomavirus HPV16 and/or HPV18 which comprises

(a) applying a polymerase chain reaction technique to a sample of humancervical tissue cells so as to amplify the amount of a selectedcharacteristic DNA portion of any such carcinogenic HPV presentcomprising the steps of:

(i) heating to dissociate the DNA strands,

(ii) adding oligonucleotide primers defining each end of saidcharacteristic DNA portion,

(iii) cooling to allow the primers to anneal to the dissociated DNAstrands,

(iv) adding a DNA polymerase,

(v) at the cooled temperature allowing formation of DNA complementary toeach strand of said characteristic DNA portion,

(vi) heating to dissociation temperature, and repeating steps (iii) to(vi), optionally omitting step (iv) where a heat stable DNA polymeraseis used.

(b) detecting the present/absence in the amplified sample of saidcharacteristic DNA portion characteristic of HPV16 or HPV18.

The invention also extends to the specific oligonucleotide primers used,and to specific labelled oligonucleotide probes, as describedhereinafter.

Embodiments of this invention will now be described by way of exampleonly.

EXAMPLES

Summary of typical technique(s) used

1. Scrapes or wart tissue are collected in the clinic by routine methodsand stored, if necessary, for up to 3 days before transport to thelaboratory for analysis.

2. Cells are lysed and their DNA denatured.

3. Viral DNA is amplified by a polymerase chain reaction.

4. Viral DNA is detected directly, e.g., by electrophoresis and staining[=end-point of test],

OR

4a. Samples (multiple) are applied to a charged nylon membrane using adot manifold, along with appropriate standards and controls.

5. Filter is prehybridized.

6. Filter is hybridized with labelled mixed viral DNA probes, onemixture containing probes for the ‘high risk’ (potentially carcinogenic)and the other probes for the ‘low risk’ HPVs.

7. Filters are washed under conditions of appropriate stringency whereonly closely related sequences will remain attached (through hydrogenbonding of the complementary DNA strands of each).

8. Autoradiography, (Black spots of exposure on the X-ray film where asample has been applied shows that it contains a HPV virus of thecategory probed for.), OR

8a. Perform nonradioactive detection method. (Coloured spot or othersignal, as appropriate, shows that it contains a HPV virus of thecategory probed for.)

Examples of oligonucleotides that work in test described

PRIMERS

For each HPV type or category two synthetic oligonucleotides aresynthesized for sue as primers in the extension and amplification stepof PCR. These oligonucleotides correspond to DNA flanking a specific“target” sequence of interest, this being a sequence of DNA in the viralgenome that is unique for HPV and differs from one HPV type or category(‘high risk’ vs ‘low risk’) to the next. We have found theoligonucleotides described below to be suitable; however, otherappropriate sequences can also be chosen for the specific viral typesindicated or for other types of papillomavirus.

Low risk HPVs

For HPV6 and HPV11 a suitable target sequence has been chosen within theE6 open reading frame of the viral genome. Suitable DNA sequencesflanking this target sequence for use as primers are as follows (whereposition number refers to the nucleotide sequence of the viral genome)and are the same for each of these HPVs:

HPV1/11 primer 1: ^(5′)ATGCCTCCACGTCTGCAAC^(3′) POSITION: 115 133

HPV6/11 primer 2: ^(3′)TACGTGACTGGTGGGCGTCTC^(5′)POSITION: 208 227

These primers have complementary sequences on HPV6 and HPV11. Theydiffer from sequences in the genomes of HPV16, HPV18 and HPV33, whereseparate primers flanking a chosen DNA target sequence are synthesizedfor these, together with oligonucleotides suitable for use as probes,using the same principal as described above and below, respectively, forHPV6 and HPV11.

High risk HPVs

For HPV16 and HPV33 suitable sequences flanking a chosen target sequenceare as follows:

HPV16/33 primer 1: ^(5′)TGAGGTATATGACTTTGCTTTT^(3′) POSITION: 223 244

HPV16/33 primer 2: ^(3′)AATTAATCCACATAAT^(5′) POSITION: 401 416

For HPV18 suitable sequences flanking a chosen target sequence are asfollows:

HPV18 primer 1: ^(5′)ACAGTATTGGAACTTACAGA^(3′) POSITION: 418 437

HPV18 primer 2: ^(3′)TTTACATATCTAAAAATAAG^(5′) POSITION: 508 527

It is most convenient to add primers for all HPV types to each sample.The sample may then be divided and probed for each category of HPV types(high risk vs low risk).

TARGET OLIGONUCLEOTIDES FOR USE AS PROBES

Low risk HPVs

Synthesize oligonucleotide (target) sequence for use as probe. Thiscorresponds to a region between the two primers, and is identical foreach of the viral types HPV6 and HPV11, but differs, of course, from anyother viral or other DNA sequence. These sequences are as follows:

HPV6/11 target oligonucleotide: ^(5′)GCAAGACGTTTAATCT^(3′) POSITION: 151166

For detection of HPV16 and HPV33 the following oligonucleotide (target)sequence is synthesized for use as a probe:

HPV16/33 target oligonucleotide: ⁵′GTGAGTATAGACATTAT^(3′) POSITION: 324340

For detection of HPV18 specifically, or within the category of high-riskHPVs, the following oligonucleotide (target) sequence is synthesized foruse as a probe:

HPV18 target oligonucleotide: ^(5′)GATTTATTTGTGGTGTATAGA^(3′) POSITION:460 480

The exact location of all of these sequences may be seen by examinationof the published sequences of the viral genomes. It should be emphasizedthat this same principle, namely synthesis of oligonucleotides that willhybridize to opposite strands of the DNA flanking a specific targetsequence in HPV described in the publications listed above and insequences of other HPVs when published.

Labelling target oligonucleotides

Radioactive labelling

5═ end label the ˜30 mers with [γ-³²P]dATP using T4 polynucleotidekinase [Berkner and Folk, J. Biol. Chem. 252: 3176-80, 1977]. A kit isavailable from Dupont (new England Nuclear) that can be used for thisstep.

i. Mix: DNA with 5′ terminal phosphates 1-50 pmoles 10 x exchangereaction buffer* 5 μl 5 mM ADP 3 μl [γ−³²P]dATP (sp. act. 3000 Cl/mmol)100 pmoles (i,e., 30 μl of a 10 mCi/ml solution) Distilled water to 50μl T4 polynucleotide kinase 1 μl (20 units) *10x Exchange reactionbuffer: 0.5 M imidazole.Cl (pH 6.6) 0.1 M MgCl₂ 50 mM dithlothreitol 1mM spermidine 1 mM EDTA ii. Incubate at 37° C. For 30 minutes. iii Add 2μl of 0.5 M EDTA. iv. Extract once with phenol/chloroform.

Nonradioactive labelling

Various methods are becoming available for nonradioactive labelling ofoligonucleotides for use as hybridization probes. These are moreappropriate for use in a HPV detection kit as they generally haveacceptably long half-lives and avoid dangers from and the need for alicense for use of radioactivity. We have used non-radioactiveoligonucleotides made by one such new technique (performed by BRESA,Adelaide) in which alkaline phosphatase is coupled directly to thetarget oligonucleotide. Alkaline phosphatase labelled oligonucleotidesof any sequence specified are available made-to-order from BRESA.

Reagents

Collection buffer=phosphate buffered saline (PBS)=140 mM NaCl, 3 mM KCl,8 mM Na₂HPO₄, 12H₂O, 1.5 mM KH₂PO₄.

Lysis buffer=50 mM NaCl, 50 mM Tris, pH 7.5, 0.5% SDS, 10 mM EDTA.

Enzyme powder=Proteinase K (concentration after addition of 10 ml lysisbuffer=50 μg/ml)

Deproteinization reagent 1=phenol/chloroform/isoamyl alcohol (25:24:1,v/v)

Deproteinization reagent 2=chloroform/isoamyl alcohol (24:1, v/v)

Extraction solution=3 M sodium acetate

PCR buffer (if using normal Klenow)=50 mM NaCl, 10 mM Tris.HCl, pH7.6,10 mM MgCl₂.

PCR buffer (if using heat stable DNA polymerase)=50 mM Tris.HCl, pH 8.8(at 25° C.), 10 mM ammonium sulphate, 10 mM MgCl₂.

PCR reagent buffer = 10 x PCR buffer .10 μl dNTPs 16 μl (4 μl of each 50mM stock) Dimethylsulphoxide 10 μl Primers 1 μl of each 500 ng/μl stock(= 4 μl in total) DNA (cells or DNA) x μl dH₂O Make volume up to 100 μl

DNA polymerase solution=1 U/μl of DNA polymerase (Klenow fragment)

Prehybridization solution=6×SSC, 25×Denhardt's solution, 0.5% sodiumdodecyl sulphate.

Hybridization solution = 6 × SSC, 1 × Denhardt's solution and 0.5% SDS,with 100 ng/ml alkaline phosphatase linked oligonucleotide probe.

Wash solution 1=6 x SSC, 0.1% SDS

Wash solution 2=6 x SSC

Wash solution 3=1 M NaCl, 0.1 M Tris.HCl, pH 9.5, 5 mM MgCl₂

Colour reagent solution=0.33 mg/ml nitroblue tetrazollium (NBT), 0.17mg/ml 5-bromo-4-chloro-3-indoyl phosphate (BCIP), 0.33% v/vdimethylformamide in 0.1 M Tris.HCl, pH 9.5, 0.1 M NaCl, 5 mM Mg Cl₂

TE=10 mM Tris.HCl, pH 8.0, 1 mM EDTA

SSC=standard saline citrate (1 x SSC=0.15 M sodium chloride, 0.015 Mtrisodium citrate, pH 7.0)

Denhardt's solution = 5 g Ficoll, 5 g polyvinylpyrrolldone, 5 g bovineserum albumin, made up to 500 ml with distilled water.

SDS=sodium dodecyl sulphate

PROTOCOL FOR CERVICAL or other anogenital, SCRAPES

Collection of specimen from patient

1. Collect scrape from the patient in the clinic using a speculum (i.e.,as for a Pap smear) or, preferably, by cervicovaginal lavage in the caseof cervical screening.

2. Place the stick with scrape in collection tube provided. (This tubecontains sterile collection buffer.)

3. The scrape tissue may be kept at 4° C. for 2-3 days or frozen at −20°C. for longer term storage.

Sample preparation

1. Take collection tube and shake off residual cells on speculum intocollection buffer in tube. Pour suspension into an Eppendorf tube.

2. Microfuge for 15 seconds. Pour off PBS from pellet. Resuspend cellsin 500 μl fresh collection buffer by vortexing briefly, Microfuge again.

3. Repeat step 2 two more times.

4. Determine number of cells per ml using a haemocytometer.

Procedure for polymerase chain reaction (Normal Klenow)

1. Add a volume of cell suspension that contains ˜10,000 cells to 35 μldistilled water in an Eppendorf tube.

2. Place tube in water bath heated to 95-98° C.

3. After 10 minutes remove tube and microfuge briefly to removecondensation.

4. Immediately add 65 μl PCR reagent buffer.

5. Place tube in a second water bath heated to 37° C.

6. After 2 min add 1 μl DNA polymerase solution; allow reaction toprocede for 2 min at 37° C.

7. Place tube back in 95-98° C. water bath for 2 minutes, then microfugebriefly.

8. Repeat steps 5-7 twenty five times.

Procedure for polymerase chain reaction (Thermophilic polymerase fromThermus aquaticus)

A DNA polymerase that is resistant to 95° C. is used instead. Use ofthis enzyme facilitates the test to some extend and, most importantly,reduces costs to ˜20%. We have used the enzyme marketed by New EnglandBiolabs and can be purified from a strain from hot springs in Rotorua,New Zealand. Protocol is as above, except 93° C. for 5 min, add 50 μlliquid paraffin to prevent evaporation, then: add 4 U polymerase andincubate in 50° C. bath for 30 s, 63° C. bath for 90 s, and 93° C. bathfor 30 s. Repeat this cycle e.g. 50 times.

Detection of viral DNA produced by PCR procedure

A number of approaches are possible from this point on. Some of those weuse are described.

A. POLYACRYLAMIDE GEL ELECTROPHORESIS FOR DIRECT VISUALIZATION OF PCRPRODUCTS

1. Pour a 12% non-denaturing polyacrylamide gel.

2. When the gel has set load half of each PCR mixture (50 μl) andelectrophorese for 2 h at 15 V/cm. Include 0.5 μg of a pBR322/HpaIIdigest as molecular weight marker.

3. After electrophoresis immerse gel in ethidium bromide solution (50μg/ml in distilled water) and allow to stain for 30 min.

4. Sample(s) positive for HPV16 or HPV33 show as a discrete band ofapproximately 200 bp. Samples positive for HPV6 or HPV11 show as adiscrete band of approximately 120 bp. Samples positive for HPV6/11 andHPV/16/33 show as discrete 200 bp and 120 bp bands.

OR

B. ALKALINE PHOSPHATASE OLIGONUCLEOTIDE PROBE ONTO DOT BLOT

1. Application of samples to membrane i) Place 25 μl of sample in anEppendorf tube. Add 25 μl of 1.0 M sodium hydroxide solution, followedby 12.5 μl of distilled water. ii) Place tube in 95-98° C. water bathfor 3 minutes. iii) Apply all of mixture to nylon membrane providedusing Hybri-dot ® manifold. iv) Rinse with 2 x SSC. v) Allow membrane todry. 2. Probing membrane with alkaline phosphatase-linkedoligonucleotide probe i) Prehybridization - Place membrane in plastic;bag provided and add 10 ml of prehybridization solution. Seal bag andincubate at 30° C. for 40 min with agitation. ii) Hybridization - Emptycontents of bag and replace with 10 ml of hybridization solution. Resealplastic bag and incubate at 30 C. for at 40 min with agitation. iii)Washing - Remove membrane from bag and place in a dish containing 500 mlof wash solution 1 preheated to 37° C.. Agitate for 10 minutes at thistemperature. Replace solution with 500 ml of wash solution 2 and agitatefor a further 10 min at 37° C.. Replace solution yet again, this timewith 200 ml of wash solution 3, and agitate for 5 min at roomtemperature. Repeat the wash solution 3 step five more times iv) Colourdevelopment - Place membrane in shallow dish containing 25 ml of colourreagent solution. Allow colour to develop for several hours or overnightat room temperature.

OR

C. RADIOACTIVE OLIGONUCLEOTIDE PROBE ONTO DOT BLOT

i) Prehybridization The membrane is prehybridized for at least 1 h at30° C.

Several approaches can be used for this.

(a) One uses a specially designed perspex block constructed in theLaboratory's mechanical workshop. The block is stood upright in a 30° C.water bath with the membrane, bathed in the following solution, inside.

(b) Another approach is to cut out two sheets of Whatman type 542 paperto dimensions slightly larger than the membrane. Next, place membrane ontop of one sheet and wet membrane with 2 ml prehybridization solution.Then place other sheet of Whatman on top and place the resulting‘sandwich’ in a plastic bag with the rest of the prehybridizationsolution.

Prehybridization mixture: (10 ml total volume) 10% (wt./vol) non fatmilk powder (e.g., Diploma ®) 0.5 ml 20x SSC (3M NaCl, 0.3 M sodiumcitrate) 2.5 ml 20% (wt./vol) SDS (sodium dodecyl sulphate) 0.5 ml 10mg/ml carrier DNA (e.g., from herring sperm) 2.0 ml Sterile distilledwater 4.5 ml

ii) Hybridization Pour off prehybridization mixture and replace withhybridization mixture, which is the same except for the addition ofradioactively labelled viral DNA (1 ng/ml hybridization mixture).

Hybridization is allowed to proceed overnight at 30° C. in the perspexblock or plastic bag.

iii) Washing membranes Membrane filters are washed under conditions ofhigh stringency. This is to remove all radioactive DNA probe except thatwhich is attached specifically, by hydrogen bonding, to identical viralDNA sequences that may be present within a sample.

iv) Autoradiography

1. The membrane is blotted with filter paper, but not allowed to dry,and while still moist, is taped to a sheet of 3MM Whatman paper, thencovered with cling wrap.

2. This is applied to a suitable x-ray film (e.g., Kodak XR-5) in adark-room and sealed in an autoradiography cassette with twointensifying screens (DuPont).

3. Autoradiography is allowed to proceed at −80° C. for 2.5-12 h.

4. Dark spots on the autoradiograph indicate the presence of HPV type.

PROTOCOL FOR WARTS AND BIOPSIES

Reagent preparation

Prepare lysis buffer by adding 1 ml distilled water to lysis bufferpowder and then mixing it with the enzyme powder in vial.

Sample preparation

1. Rinse tissue 3 times in 500 μl collection buffer (as above forscrapes).

2. Cut tissue into ˜1 mm pieces.

3. Add to tissue 700 μl lysis buffer enzyme solution.

4. Leave at 37° C. until tissue appears completely digested or overnightat 37° C.

5. Add 350 μl deproteinization reagent 1 and 350 μl deproteinizationreagent 2. Mix vigorously.

6. Microfuge for 15 seconds and remove top layer into a fresh tube(discard lower phase).

7. Repeat steps 5 and 6 three times.

8. Add 700 μl of deproteinization reagent 2, mix and microfuge for 15seconds.

9. Remove top layer into a fresh tube and add 100 μl of extractionsolution. Immediately add 1,4 ml of ice-cold absolute ethanol. Place at−20° C. for 1 hour or −80° C. for 15 minutes.

10. Microfuge for 10 minutes and then pour off ethanol carefully. Allowpellet (=DNA) to dry for 15 minutes.

11. Resuspend DNA in 50 ml of TE and, if possible, determine DNAconcentration at 260 nm using a spectrophotometer. (1 O.D.₂₆₀ unit=50μg/ml).

Procedure for polymerase chain reaction (PCR)

1. Add a volume of DNA solution that contains ˜1 μg of DNA to 100 μl ofPCR reaction mixture in an Eppendorf tube.

2. Place tube in water bath heated to 95-98° C.

3. After 10 min microfuge tube briefly (−5 seconds) to removecondensation.

4. Place tube in water bath preheated to 37° C.

5. After 2 min add 1 μl of DNA polymerase solution and continue 37° C.incubation for 2 min.

6. Place tube back in 95-98° C. water bath for 2 min.

7. Repeat steps 4-6 twenty five times.

Detection of viral DNA produced by PCR procedure

See as above for scrapes protocols.

Standards and controls

Standards: Pure viral DNA of HPV6, HPV11, HPV16, HPV18 and HPV33 oroligonucleotides corresponding to at least the hybridizing target regionof each HPV are added to the membrane in amounts of 125, 12.5, 1.25 and0.125 pg for complete HPV or correspondingly less for oligonucleotides.

Controls:

i) Neonatal foreskin DNA (detects the unlikely occurrence of anynonspecific hybridization to human skin DNA) in amounts of 10 ng, 50 ng,100 ng and 2 μg is applied to each membrane.

ii) Positive control: DNA from HPV6/11 and HPV16/18/33 infected samples.

iii) Alu-repeated sequence DNA (to hybridize to the Alu probe) inamounts of 10 pg, 50 pg, 100 pg and 2 ng is applied to each membrane.(Optional)

Automation

We have designed and constructed a working prototype machine forperforming the PCR reaction automatically.

Total Time for Test Result (After receipt of samples in lab.)

Several hours-2 days.

Results Obtained with the Test

Results have indicated the importance of direct HPV detection in patientevaluation. For example, many patients that had normal cytology ofcervical cells were found to be infected with the carcinogenic varietiesof HPV. This may reflect the fact that our test can detect infection atan earlier stage, perhaps even before the virus has had a change tocause a morphological change in the cell. Moreover, the lesionsassociated with the potentially carcinogenic varieties of HPV are oftenflat, rather than the large obvious condylamas caused by the more benignvarieties, and are therefore not as easily seen in the clinic.

SPECIFIC EXPERIMENTS REPRESENTING EXAMPLES OF METHOD IN OPERATION

Drawings are given in the figures in lieu of photographs as thephotocopying process gives poor resolution.

A. PCR amplification of pure HPV16 viral DNA using normal Klenow. 1 ngHPV16/pBR322 was subjected to 20 rounds of: 95° C. for 2 min (stranddissociation), then 37° C. for 2 min (annealing), then 1 U Klenow enzymeadded and 37° C. incubation for 2 min. Half (50 μl) of the resultingmixture was electrophoresed on a 2% agarose gel and the result is shownin FIG. 1. Amount=2.5×100 ng=250 ng. Region amplified={fraction(1/40)}th of total HPV16 sequence. Total amplification=250×40=10,000 x,(i.e., efficiency of reaction was 60% for each cycle).

B. PCR amplification of wart biopsy DNA with HPV16/33 test. The DNA ofanal wart Gap289 was phenol/chloroform extracted and ethanolprecipitated. 1 μg of DNA was then subjected to 23 rounds ofamplification (each: 95° C. for 2 min, then 37° C. for 2 min, 1 U Klenowadded and 37° C. for 2 min). 40% (50 μl) was electrophoresed on a 2%agarose gel. Ethidium bromide stained DNA is shown in FIG. 2. The PCRsample and standards were spotted onto a membrane and hybridizationperformed overnight with ³²P-labelled HPV16/33 target oligonucleotideprobe. Wash conditions were 22° C. for 5 min, then 38° C. for 10 min in5 x SSPE/0.1% SDS. Result of autoradiography is shown in FIG. 3. Notethat amounts are for recombinant viral DNAs used, only a fraction ofwhich represents amplified sequence, within which is the targethybridization region.

C. PCR amplification of pure HPV16 recombinant viral DNA using normalKlenow, 1 ng of HPV16/pBR322, 10 mM Tris.HCl, pH 7.6, 10 mM MgCl₂, 50 mMNaCl 500 ng of each of the HPV16/33 primers, 4 μl of each dNTP (50 mMstock). 23 rounds of: 98° C. for 2 min, then 37° C. for 2 min, addKlenow, then 37° C. for 2 min. Electrophoresis on 2% agarose gel.Ethidium bromide stained gel is shown in FIG. 4. Lanes 1 and 3 are DNAsize markers, viz. pBR322 cut with HpaII and SPP-1, respectively. Asingle DNA band of the expected size (˜200 base pairs) can be seen inlane 2. To prove this Southern blotting was then performed on this gel,using for hybridization conditions: 5 x SSPE, 2% SDS, 0.5 ml BLOTTO, 0.5ml of 10 mg/ml salmon sperm DNA, 5.5 ml distilled water.Prehybridization was for 1 h at 30° C. and hybridization was overnightat 30° C. in the above solution with ˜1 ng/ml of end-labelled targetHPV16/33 oligonucleotide probe. Washing was for 10 min at 38° C. in 1 xSSPE, 0.1% SDS. Result in FIG. 5, showing massive hybridization to aband at the expected position in lane 2 (i.e., at the same position asthe band of stained DNA in FIG. 4 lane 2).

D. Biopsies of anal warts. Patient specimens: Gap119, Gap278, Km,Gap289, 11912C. Conditions as described above in C. FIG. 6 is anautoradiograph after radioactive probing: Km and Gap289 were +ve, i.e.,contained HPV16.

E. Southern blot of biopsies +ve for HPV16. This experiment shows thatthe HPV16/33 oligo probe hybridizes to the correct restriction fragmentsin unamplified biopsies and pure viral DNA; also that there is no crosshybridization to other HPV types.

Lane 1—8 μg Gap289 cut with BamHI/PstI

Lane 2—blank

Lane 3—100 ng HPV33 cut with BgIII/PstI

Lane 4—100 ng HPV16 cut with BamHI/PstI

Lane 5—100 ng HPV18 cut with EcoRI/XbaI

Lane 6—100 ng HPV11 cut with BamHI/PstI

Lane 7—100 ng HPV6 cut with EcoRI/PstI

The autoradiograph (shown diagrammatically in FIG. 7) showshybridization of radioactive HPV16/33 oligo probe to HPV16 in lane 4 andto HPV33 in lane 3. No hybridization occurred to HPVs 6, 11 and 18 inlanes 7, 6 and 5, respectively, showing that the probe is specific forHPVs 16 and 33. Hybridization in lane 1 shows that specimen Gap289contained HPV16.

F. Cervical scrapes—HPV16/33 PCR amplification. This shows the techniquecan detect specific HPV in scrape specimens. Scrapes: Pt and Mo. ˜10,000cells were suspended in 35 μl water, heated to 98° C. for 10 min, 65 μlPCR mixture added and normal protocol followed with 30 rounds ofamplification using normal Klenow. Samples were dotted onto membranesand probed with radioactive oligo probe. Result (FIG. 8): Pt scrape was+ve with HPV16/33 probe, Mo was −ve.

G. Souther blot of scrape. This was to confirm that Pt scrape had givenreal amplification, (i.e., is proof of validity of result in H.) Gelused was 1.5% agarose. In FIG. 7:

Lane 1—25 μl Pt PCR digested with Bg/I/BamHI in PCR buffer.

Lane 2—40 ng of HPV33 insert.

Lane 3—40 ng of HPV16 insert.

Lane 4—0.5 μg of pBR322 digested with HpaII.

Lane 5—Bacteriophage λdigested with HindIII.

FIG. 9 depicts the stained gel result. FIG. 10 shows the result ofradioactive target oligo probing. FIG. 11 shows the result of alkalinephosphatase target oligo probing. A dark band of hybridization can beseen at the correct position in lane 1 for the patient specimen's PCRproducts and bands in lanes 2 and 3 corresponding to the position of thewhole virus. This confirms the presence of HPV16 in the cervical scrape.

The following examples (H-J) show additional positive results with thetechnique used on various biopsies and scrapes.

H. Various scrapes and anogenital wart biopsies—HPV6/11 PCR. In FIGS. 12and 13:

Lane 1—0.4 μg pBR322 cut with HpaII.

Lane 2—0.9 μg F9885 wart DNA, HPV/6/11 oligos only.

Lane 3—1 μg Ow wart DNA, HPV6/11 and HPV16/33 oligos.

Lane 4—3 μl cells from F11912 cervical scrape, HPV6/11 oligos only.

Lane 5—3 μl cells from F11912 vaginal scrape, HPV6/11 oligos only.

Lane 6—5 μl cells from F11912 rectal scrape, HPV6/11 oligos only.

30 rounds were performed under normal protocol conditions, 50 μl of eachPCR mixture was loaded on a 12% non-denaturing polyacrylamide gel. Thestained gel is shown in FIG. 12. (The band at ˜34 bp is an artifactassociated with the HPV6/11 primers.) This membrane was electroblottedat 20V/100 mA for 1 h and probed with the radioactive HPV6/11 oligoprobe. Result of 2 h exposure to x-ray film is shown in FIG. 13. Bandsin lanes 4 and 5 indicate that cervical and vaginal, but not rectalcells from this patient were infected with HPV6/11.

I. Warts—HPV6/11 PCR. Results are shown in FIG. 14:

Lane 1—pBR322 cut with HpaII

Lane 2—750 ng of F8408 labial wart

Lane 3—1 μg Gap252 anal wart

Lane 4—860 ng Tr anal wart

Lane 5—750 ng Bi anal wart

(Conditions: 25 rounds, HPV6/11 primers only; 50 μl of each PCR mix runon gel.)

J. Cervical warts—HPV6/11 and HPV16/33 primers. 50 μl run on 12%polyacrylamide gel, results as shown in FIG. 15.

Lane 1—pBR322 cut with HpaII

Lane 2—˜500 ng Hn biopsy

Lane 3—˜500 ng Hs biopsy

Lane 4—˜500 ng Ls biopsy

K. Cervical scrapes—HPV6/11 and HPV16/33. This example shows thatprimers for different HPVs can be used together in the same reactionmixture to generate amplification products unique for each HPV type.Results are shown in FIG. 16.

Lane 1—pB322 cut with HpaII.

Lane 2—1 ng of HPV6/pAT153 and 1 ng of HPV16/pBR322; HPV6/11 andHPV16/33 primers.

Lane 3—3 μl cells of CSC019 scrape. HPV6/11 and HPV16/33 primers.

Lane 4—3 μl cells of CSC328 scrape. HPV6/11 primers.

Lane 5—3 μl cells of Pt scrape, HPV6/11 primers.

(Conditions: 25 rounds, normal protocol conditions.) In lane 2 of FIG.16 one can see the 200 bp HPV16/33 amplification product (faint) and the120 bp HPV6/11 product (darker). Lanes 3-5 were negative (only primerbands seen), i.e., did not contain HPV6/11.

L. Scrapes and biopsies—HPV16/33 PCR. This experiment is a comparison ofradioactive and nonradioactive probing for the detection of HPVs by thePCR method. In FIG. 12:

Lane 1—pBR322 cut with HpaII

Lane 2—Blank

Lane 3—HPV6/pAT153 and HPV16/pBR322 PCR from 1

Lane 4—15 μl of Km PCR

Lane 5—15 μl of Gap289 PCR

Lane 6—15 μl Pt scrape PCR

Gel (12% polyacrylamide) was run and stained (FIG. 17), thenelectroblotted and probed with alkaline phosphatase (FIG. 18), andradioactive (FIG. 19) HPV16/33 oligo probe. In FIG. 17 the ˜200 bp bandindicative of HPV16/33 can be seen in lanes 6, 5, 4, and 3 and the 120bp HPV6/11 band can be seen in lane 3. In FIG. 18 a single band ofhybridization to the ˜200 bp HPV16/33 amplification products can be seenin each of lanes 6-3. FIG. 19 shows the result of a 1.5 h exposure tothe x-ray film: hybridization can be seen to the ˜200 bp HPV16/33amplification products. FIG. 20 is a dot blot probed with radioactivetarget oligo probe (Right: diagram of position of samples on dot blot;Left: result of hybridization).

M. Heat stable polymerase—HPV6 PCR. 1 ng HPV6/pAT153, 2 units of Thermusaquaticus DNA polymerase added initially and after every 10 rounds for40 rounds. The conditions used were exactly as specified in the NewEngland Biolabs protocol. The result (FIG. 21) demonstrates that theheat stable polymerase is also able to produce the desired amplificationproducts. In this figure:

Lane 1—pBR322HpaII

Lane 2—40 μl of HPV6/pAT153 PCR

N. Heat stable polymerase—HPV16 and biopsy. After amplificationHPV16/pBR322 cut with HinfI should give 2 fragments of 52 bp and 142 bp.5 ng HPV33/plink and 1 μg Gap289 were subjected to 40 rounds with heatstable polymerase (lanes 4 and 5 in FIG. 22). The conditions used were:50 mM Tris.HCl, pH 8.8 at 25° C., 10 mM MgCl₂, 10 mM (NH₄)₂SO₄, 10 μlDSMO, 500 ng of each HPV16/33 primer, 4 μl of each dNTP (50 mM stock),and 50 μl liquid paraffin, 4 U DNA polymerase was added at the start.Incubation was in 93° C. incubator for 30 s, then 50° C. for 30 s, then63° C. for 45 s. In FIG. 22:

Lane 1—pBR33/HpaII

Lane 2—HPV16/pBR322 cut with HinfI (20 μl) PCR+2 μl 10 x HinfI buffer+1μl enzyme+6 μl dH₂O)

Lane 3—20 μl of HPV16/pBR322 PCR

Lane 4—20 μl of HPV33/plink PCR

Lane 5—20 μl of Gap289

O. Diluted biopsy. 100 PCR rounds using heat stable polymerase. Wartbiopsy Gap289 (1 μg/μl) was diluted 1:10³, 1:10⁶ and 1:10⁹ and 100rounds of amplification were performed using 1 μl of each dilution and 4U of heat stable DNA polymerase initially, then an additional 2 U addedafter 50 rounds. Other biopsies and scrapes were subjected to 50 roundsonly. Conditions were as in example P. The result shows the extremesensitivity of the test: the best results with this excessive number ofrounds of amplification were obtained with the most dilute sample. FIG.23 shows the ethidium bromide stained DNA gel. The gel waselectroblotted and probed with radioactively labelled HPV6/11 (FIG. 24)and HPV16/33 (FIG. 25) target oligo probe. The result shows the extremespecificity of the test. In FIG. 15(b) it can be seen that only in lane5 and 6 where HPV6/11 primers were used was there hybridization to a 120bp band, the size of the HPV6/11 amplification product. Absolutely nohybridization was seen in the lanes where samples had been amplifiedusing only HPV16/33 primers. In FIG. 15(c) hybridization to the ˜200 bpband of HPV16/33 amplification products can be seen for the biopsy(lanes 2-4) and the anal scrape (lane 5). In FIGS. 23, 24 and 25 thelanes are as follows:

Lane 1—pBR322 cut with HpaII

Lane 2—Gap289 (1:10³) PCR (HPV16/33 primers only)

Lane 3—Gap289 (1:10⁶) PCR (HPV16/33 primers only)

Lane 4—Gap289 (1:10⁹) PCR (HPV16/33 primers only)

Lane 5—Anal scrape Gap402 PCR (HPV6/11 and HPV16/33 primers)

Lane 6—Bs wart PCR (HPV6/11 and HPV16/33 primers)

Lane 7—Cervical scrape 11912 (HPV16/33 primers only)

Lane 8—Vaginal scrape 11912 (HPV16/33 primers only)

P. Hybridization of target oligo probe to unamplified HPV16, HPV33 andbiopsies. Ordinary restricted HPVs and biopsies were run on a gel andstained (FIG. 26), then probed with HPV16/33 target oligo probe. Resultis shown in FIG. 27, where hybridization can be seen in each case (lanes2-5) and to correct restriction fragment in the case of recombinantviral DNA. Six days of exposure to x-ray film were required for thisunamplified DNA. In FIGS. 26 and 27:

Lane 1—Bacteriophage λcut with HindIII/EcoRI

Lane 2—5 μg Bs wart DNA, Bg/II/BamHI

Lane 3—5 μg Gap289, Bg/II/BamHI

Lane 4—HPV16/pBR322, BamHI

Lane 5—HPV33/plink, Bg/II

Lane 6—SPP-I

Q. Dot blots showing hybridization of HPV16/33 alkaline phosphataseoligo probe to HPV16 are shown in FIG. 28.

R. HPV18 PCR. 30 rounds with normal Klenow DNA polymerase Result isshown in FIG. 29, where the lanes are:

Lane 1—HPV16 insert PCR products; HPV16/33 primers.

Lane 2—HPV18 insert PCR products; HPV18 primers used.

The HPV18 PCR products of ˜100 bp can be seen in lane 2. In lane 1 arethe HPV16 PCR products of ˜200 bp.

What is claimed is:
 1. A method for distinguishing between high riskstrains 16 and 33 or 18 of human papillomavirus and low risk strains 6and 11 of human papillomavirus in a sample of human cervical tissuecomprising: a) obtaining DNA from the human cervical tissue sample, b)simultaneously or individually adding oligonucleotide primer pairs whichare specific for the E6 region of i) HPV 16 and 33, ii) HPV 18 or iii)HPV 6 and 11, c) amplifying the HPV E6 region by polymerase chainreaction using the E6 specific primer pair(s) and d) detecting thepresence or absence of amplification products specific for HPV16 and 33or 18 or HPV 6 and 11 to thereby distinguish between the presence ofhigh risk strains HPV 16 and 33 or 18 and low risk strains HPV 6 and 11.2. A method according to claim 1 wherein the amplification products aredetected using a labeled oligonucleotide hybridization probe.
 3. Amethod according to claim 2 wherein the oligonucleotide probe is labeledwith a ³²P radioactive label.
 4. A method according to claim 2 whereinthe oligonucleotide probe is labeled with an alkaline phosphatase enzymelabel.